Novel lactobacillus plantarum isolated from leaves of camellia sinensis

ABSTRACT

Disclosed are novel  Lactobacillus plantarum  strains as well as a composition containing the novel  Lactobacillus plantarum  strains or a culture thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/670,068, filed on Mar. 26, 2015, which is a divisional of U.S. patentapplication Ser. No. 14/124,483, filed on Dec. 6, 2013, which claimspriority to PCT Application No. PCT/KR2012/004569, filed on Jun. 8,2012, which claims priority to Korean Patent Application No.10-2011-0056465, filed on Jun. 10, 2011, Korean Patent Application No.10-2011-0056466, filed on Jun. 10, 2011, Korean Patent Application No.10-2011-0056468, filed on Jun. 10, 2011, and Korean Patent ApplicationNo. 10-2011-0056469, filed on Jun. 10, 2011, and all the benefitsaccruing therefrom under 35 U.S.C. §119, the contents of which in itsentirety are herein incorporated by reference.

TECHNICAL FIELD

The present disclosure relates to a novel Lactobacillus plantarumisolated from tea tree (Camellia sinensis) leaves.

BACKGROUND ART

Tea for drinking is one obtained by deactivating oxygenase present inshoots or leaves of theaceous Camellia sinensis and removing watertherefrom. It contains caffeine, tannin, flavonoids, essential oils andthe like as well as vitamins, and has been widely used at various fieldssuch as a food field.

DISCLOSURE Technical Problem

The present disclosure is directed to providing a novel Lactobacillusplantarum strain. Further, it is directed to providing a compositionincluding the novel Lactobacillus plantarum strain or a culture solutionthereof.

Technical Solution

In one aspect, there is provided a Lactobacillus plantarum APsulloc331261 strain.

In one aspect, there is provided a Lactobacillus plantarum APsulloc331263 strain.

In one aspect, there is provided a Lactobacillus plantarum APsulloc331266 strain.

In one aspect, there is provided a Lactobacillus plantarum APsulloc331269 strain.

In another aspect, there is provided a composition including at leastone of the Lactobacillus plantarum APsulloc 331261 strain, the APsulloc331263 strain, the APsulloc 331266 strain and the APsulloc 331269strain, or a culture solution thereof.

Advantageous Effects

The novel Lactobacillus plantarum strain according to the presentdisclosure is excellent in acid resistance, and therefore, it is alsoviable in the stomach when it is taken by a food, and further, it mayhave higher intestinal deliverity. The Lactobacillus plantarum strainaccording to the present disclosure is excellent in bile acidresistance, and therefore, it is superior to intestinal fix. Further,the Lactobacillus plantarum strain according to the present disclosureis excellent in antibacterial activity, and therefore, it is superior toharmful bacteria inhibitory effect. The Lactobacillus plantarum strainaccording to the present disclosure has lower D-lactic acid ratio in theproduced lactic acid than the existing Lactobacillus plantarum.Accordingly, even adults or infants, who are susceptible to lactic acid,may freely intake the strain. The Lactobacillus plantarum strainaccording to the present disclosure produces fewer amount of lactic acidthan the existing Lactobacillus plantarum, and therefore, when foods arefermented by using thereof, the foods may have gentle flavor. Thus, thenovel Lactobacillus plantarum strain according to the present disclosuremay be widely used in various fields, for example, a food field.

BEST MODE

Lactic acid bacteria are bacteria producing lactic acid by degradingsugar such as glucose. The lactic acid produced by lactic acidfermentation of lactic acid bacteria may prevent growth of pathogens andharmful bacteria, and this characteristic is used for producing foodssuch as dairy foods, kimchis, brewed foods and the like. Further, thelactic acid bacteria live in the intestine of mammals and inhibitabnormal fermentation by various germs, and therefore, those areimportant bacteria useful as a medicine for intestinal disorders.

Lactobacillus plantarum is a strain belongs to the lactic acid bacteria,and it is known to mainly grow when kimchi is much fermented and tastessour. Optical isomers of the produced lactic acid are D-type and L-type.The Lactobacillus plantarum may be widely used to various foods needfermentation. Accordingly, if a Lactobacillus plantarum having excellentacid resistance, bile acid resistance and antibacterial activity isdeveloped, it may be usefully used.

Hereinafter, the present disclosure now will be described in detail.

One aspect of the present disclosure provides a Lactobacillus plantarumAPsulloc 331261 (Accession No.: KCCM11179P) strain. One aspect of thepresent disclosure provides a Lactobacillus plantarum APsulloc 331263(Accession No.: KCCM11180P) strain. One aspect of the present disclosureprovides a Lactobacillus plantarum APsulloc 331266 (Accession No.:KCCM11181P) strain. One aspect of the present disclosure provides aLactobacillus plantarum APsulloc 331269 (Accession No.: KCCM11182P)strain.

At least one of the Lactobacillus plantarum APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269 accordingto the present disclosure is a strain isolated from tea tree (Camelliasinensis) leaves, and belongs to Lactobacillus plantarum. Specifically,the Lactobacillus plantarum strain according to the present disclosuremay be isolated by a method containing: a step of salting the tea treeleaves in salt of 5 to 15 wt %, based on the weight of the tea treeleaves; a step of mixing the salted tea tree leaves with a sugarsolution, for example, 0.1% to 3% fructooligosaccharide, followed byculturing at 25 to 35° C. for 1 to 5 days; and a step of collecting thesolution cultured to less than pH 5, followed by culturing at anaerobiccondition of 25 to 35° C. for 1 to 5 days.

At least one of the Lactobacillus plantarum APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269 accordingto one aspect of the present disclosure has excellent acid resistance.When intaking lactic acid bacteria, for example, Lactobacillus plantarumas a probiotic, it is preferred to have higher intestinal deliverity, inorder to display characteristic effects of the lactic acid bacteria. Inorder to enhance the intestinal deliverity, viability in the stomach,where pH is low due to gastric acid secretion, should be high. It isknown that pH of the empty stomach may be about 1.2 to 2, but whentaking foods, pH may be about 2 to 3. The Lactobacillus plantarum strainaccording to the present disclosure may have excellent acid resistanceat more than pH 2 to 4, specifically more than pH 2 to 3.5 or pH 2.5 to4, more specifically pH 2.5 to 3.5, further more specifically about pH2.5 to 3. On the other hand, when intaking foods, it is known that theaverage retention time of the foods may be about 1 to 3 hours. TheLactobacillus plantarum strain according to the present disclosure mayhave excellent acid resistance for 0.5 to 5 hours, specifically for 1 to4 hours, at more than pH 2 to pH 4, specifically more than pH 2 to pH3.5 or pH 2.5 to 4, more specifically pH 2.5 to 3.5, further morespecifically pH 2.5 to 3. Thus, the Lactobacillus plantarum strainaccording to the present disclosure, which is viable even at lower pH ofthe stomach during the time stayed in the stomach, may have highintestinal deliverity when it is taken as a food.

The Lactobacillus plantarum strain according to one aspect of thepresent disclosure has excellent bile acid resistance. The foods passedthrough the stomach are delivered into the intestine, and at this time,the secreted bile acid helps food digestion. It is known that the strainhaving high bile acid resistance has good intestinal fix. TheLactobacillus plantarum strain according to one aspect of the presentdisclosure having excellent bile acid resistance is superior to theintestinal fix.

The Lactobacillus plantarum strain according to one aspect of thepresent disclosure has lactic acid producing ability. In general, thelactic acid produced by lactic acid bacteria may be L-type and D-type.Of them, the metabolic rate of the D-lactic acid in the body is lowerthan the L-lactic acid. Accordingly, the higher blood D-lactic acidconcentration may cause lactic acid toxicosis. Thus, it is preferredthat adults or infants, who are susceptible to lactic acid, do not takelactic acid bacteria, which largely produces D-lactic acid, if possible.

The Lactobacillus plantarum strain according to another aspect of thepresent disclosure may produce lactic acid containing D-type in anamount of 75% or less, specifically 70% or less, more specifically 65%or less.

The Lactobacillus plantarum strain according to further another aspectof the present disclosure may produce lactic acid in an amount of 17.5g/L or less, specifically 17 g/L or less, more specifically 16.5 g/L orless, further more specifically 15 g/L or less, further morespecifically 14.5 g/L or less, further more specifically 14 g/L or less,further more specifically 13.5 g/L or less.

Like this, because the Lactobacillus plantarum strain according to thepresent disclosure produces lactic acid containing D-type with lowerrate than the existing Lactobacillus plantarum, adults or infants, whoare susceptible to lactic acid, may freely take it. Further, because itslactic acid yield is lower than the existing Lactobacillus plantarum,when fermenting foods by using thereof, the foods may have gentlerflavor.

One aspect of the present disclosure provides an extract or a culturesolution of at least one of the Lactobacillus plantarum APsulloc 331261strain, the APsulloc 331263 strain, the APsulloc 331266 strain and theAPsulloc 331269 strain. Another aspect of the present disclosureprovides a composition containing at least one of the Lactobacillusplantarum APsulloc 331261 strain, the APsulloc 331263 strain, theAPsulloc 331266 strain and the APsulloc 331269 strain, an extractthereof, or a culture solution thereof.

One aspect of the present disclosure provides a food compositioncontaining at least one selected from the Lactobacillus plantarumAPsulloc 331261 strain, the APsulloc 331263 strain, the APsulloc 331266strain and the APsulloc 331269 strain, its extract, or its culturesolution.

The food composition may be a health food composition, and also may be afermented food composition, which needs fermentation, for example, teas,dairy goods, kimchis, brewed foods.

The formulation of the food composition is not particularly limited, butfor example, it may be formulated into tablet, pill, hard or softcapsule, granule, drink, caramel, diet bar, tea bag and the like. Thehealth food composition may further comprise, in addition to the activeingredient, commonly used other ingredients, which may be suitablyselected by those skilled in the art depending on the formulation orpurpose of the composition. The addition of the other ingredients maygive a synergic effect.

Determination of the dose of the active ingredient is within the levelof those skilled in the art. For example, its daily dose may beLactobacillus plantarum about 10⁵ to 10¹³ CFU/day, more specificallyabout 10⁶ to 10¹⁰ CFU/day, but is not limited thereto, and may be variedwith various factors including the age, physical condition,complication, etc. of a subject to be treated.

One aspect of the present disclosure provides a cosmetic compositioncontaining at least one of the Lactobacillus plantarum APsulloc 331261strain, the APsulloc 331263 strain, the APsulloc 331266 strain and theAPsulloc 331269 strain, an extract thereof, or a culture solutionthereof. The cosmetic composition may be provided as any formulationsuitable for topical application. For example, it may be provided as aformulation of oil in water emulsion, water in oil emulation,suspension, solid, gel, powder, paste, foam or aerosol composition. Thecomposition of the said formulation may prepared by a conventionalmethod in the art.

The cosmetic composition may further include other ingredients, whichmay not adversely affect a main desired effect, preferably may provide asynergic effect to the main effect. The cosmetic composition accordingto the present disclosure may include a material selected from the groupconsisting of vitamin, polymer peptide, polysaccharide and spingolipid.Further, the cosmetic composition according to the present disclosuremay include moisturizing agent, emollient, surfactant, UV absorbingagent, preservative, disinfecting agent, antioxidant, pH modifier,organic and inorganic dye, aromatic, cooling agent or antiperspirant.The amount of the ingredients may be suitable selected in such a mannerthat they may not adversely affect the purpose and effect of the presentdisclosure, and the amount may be 0.01 to 5 wt %, specifically 0.01 to 3wt %, based on the total weight of the composition.

One aspect of the present disclosure provides a pharmaceuticalcomposition containing at least one of the Lactobacillus plantarumAPsulloc 331261 strain, the APsulloc 331263 strain, the APsulloc 331266strain and the APsulloc 331269 strain, an extract thereof, or a culturesolution thereof. The pharmaceutical composition may be used forpreventing or treating intestinal disorder such as irritable bowelsyndrome, constipation and diarrhea.

The pharmaceutical composition according to one aspect of the presentdisclosure may be administered orally or parenterally, e.g., rectally,topically, transdermally, intravenously, intramuscularly,intra-abdominally, subcutaneously, etc. Examples of formulations fororal administration include tablet, pill, soft or hard capsule, granule,powder, fine granule, solution, emulsion, pellet and the like, but notlimited thereto. Examples for formulations for parenteral administrationinclude solution, suspension, lotion, gel, injectable solution, drop,suppository, patch or spray, but not limited thereto. The formulationmay be easily formulated according to conventional methods, andsurfactants, excipients, wetting agent, emulsifier, suspending agent,salt or buffer for osmotic pressure control, colorant, flavoring agent,stabilizer, preservative, preserved agent or other conventionaladditives may be suitably used for the formulation.

Active ingredient of the pharmaceutical composition according to oneaspect of the present disclosure may be varied with the age, sex andbody weight of a subject to be treated, pathological condition betreated, severity of the pathological condition, administration routeand the judgment of a prescriber. Determination of the dose consideringthese factors is within the level of those skilled in the art. Forexample, the daily dose may be 0.1 mg/kg/day to 5000 mg/kg/day,specifically 50 mg/kg/day to 500 mg/kg/day, but not limited thereto.

The Lactobacillus plantarum APsulloc 331261, the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 are deposited at Korean CultureCenter of Microorganisms (KCCM, address: 361-221, Yurim B/D.hongje-1-dong, Seodaemun-gu, Seoul, Republic of Korea) on Mar. 28, 2011under Accession Nos.: KCCM11179P, KCCM11180P, KCCM11181P and KCCM11182P.

(1) Lactobacillus plantarum APsulloc 331261

Depository Institution Name: Korean Culture Center of MicroorganismsAccession No.: KCCM11179P Deposition Date: 2011 Mar. 28

(2) Lactobacillus plantarum APsulloc 331263 Depository Institution Name:Korean Culture Center of Microorganisms

Accession No.: KCCM11180P Deposition Date: 2011 Mar. 28

(3) Lactobacillus plantarum APsulloc 331266

Depository Institution Name: Korean Culture Center of MicroorganismsAccession No.: KCCM11181P Deposition Date: 2011 Mar. 28

(4) Lactobacillus plantarum APsulloc 331269

Depository Institution Name: Korean Culture Center of MicroorganismsAccession No.: KCCM11182P Deposition Date: 2011 Mar. 28

Hereinafter, isolation methods, identification methods andcharacteristics of the novel Lactobacillus plantarum strains accordingto the present disclosure now will be described in detail with referenceto the examples (and experiments). However, the following examples (andexperiments) are for illustrative purposes only and not intended tolimit the scope of this disclosure.

Example 1 Isolation of Lactobacillus Plantarum Strain

Tea tree leaves 200 g are washed twice with distilled water to removeimpurities. Moisture is cleared off from the washed tea tree leaves.Then, the tea tree leaves are mixed with table salt of 8 wt %, based onthe weight of the tea tree leaves, and then stored at room temperaturefor 3 hours. The salted tea tree leaves are mixed with 1%fructooligosaccharide solution 1000 mL, and then incubated in anincubator at 32° C. for 3 days. 3 days later, whether pH of the culturedsolution is lowered to less than 5 is checked, and in the case of lessthan pH 5, the cultured solution is collected and incubated in DifcoLactobacilli MRS Agar® medium. At this time, the incubation is conductedin a 32° C., anaerobic chamber for 2 days, and then white colonies arecollected.

Through the method described above, Lactobacillus plantarum APsulloc331261, APsulloc 331263, APsulloc 331266 and APsulloc 331269 areisolated from tea tree leaves, respectively.

Example 2 Identification of Lactobacillus Plantarum Strain (1) StrainCulture

The APsulloc 331261 isolated in Example 1 is streaked on a MRS agarplate, and cultured at 37° C. for 2 days. The obtained single colony isinoculated to MRS broth 10 mL, and then cultured at 37° C. overnight toprepare a Lactobacillus plantarum strain culture solution. The methoddescribed above is repeated to the APsulloc 331263, the APsulloc 331266and the APsulloc 331269, respectively, to prepare Lactobacillusplantarum strain culture solutions.

(2) Analysis of Sugar Fermentation Pattern of Lactobacillus PlantarumStrain

The APsulloc 331261 strain culture solution prepared as described in (1)is inoculated to MRS broth 10 mL to the concentration of 0.5% andcultured at 37° C. overnight. The culture solution is centrifuged at8,000 rpm for 5 min, supernatant is removed, and then only bacteria arecollected. Then, 0.85% saline buffer 2 mL is added to the bacteria andsuspended. Later process is conducted by using API 50CHL kit(Biomerieux) according to a manufacturer's protocol. Specific process isas follows.

First of all, while gradually adding the strain suspension to APIsuspension medium 5 mL, the amount of suspension needed to makecloudiness of about McFarland Standard 2 (Biomerieux) is measured. Twiceof the measured amount of the suspension is added to API 50CHL medium 10mL, and then shaken for mixing. The above mixture is added to cupulescontaining different substrate, one drop of mineral oil is put thereto,and then the mixture is cultured at 37° C. for 2 days to analyze sugarfermentation pattern. The method described above is repeated to culturesolutions of the APsulloc 331263, the APsulloc 331266 and the APsulloc331269 strain to analyze sugar fermentation pattern.

The results of sugar fermentation patterns of the APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269, comparedwith Lactobacillus plantarum strain (KCTC3108) as a standard strain, andthe results of identification of the APsulloc 331261, the APsulloc331263, the APsulloc 331266 and the APsulloc 331269 by using the aboveresults are as shown in the following Tables. Table 1: APsulloc 331261,Table 2: APsulloc 331263, Table 3: APsulloc 331266 and Table 4: APsulloc331269.

TABLE 1 APsulloc APsulloc KCTC3108 331261 KCTC3108 331261 Substrate 24 h48 h 24 h 48 h substrate 24 h 48 h 24 h 48 h Control − − − −Esculin + + + + Glycerol − − − − Salicin + + + + Erythritol − − − −Cellobiose ? + + + D-Arabinose − − − − Maltose + + + +L-Arabinose + + + + Lactose + + + + Ribose + + + + Melibiose + + + +D-Xylose − − − − D-Saccharose + + + + L-xylose − − − − (Sucrose)Adonitol − − − − Trehalose + + + + β-Methyl- − − − − Inulin − − − −D-xylose Melezitose + + + + Galactose + + + + Raffinose − − + +Glucose + + + + Amidon − − − − Fructose + + + + (Starch) Mannose + + + +Glycogen − − − − Sorbose − − − − Xylitol − − − − Rhamnose − − − −Gentiobiose − − + + Dulcitol − − − − D-Turanose + + + + Inositol − − − −D-Lyxose − − − − Mannitol + + + + D-Tagatose − − − − Sorbitol + + + +D-Fucose − − − − α-Methyl-D- ? + − − L-Fucose − − − − mannosideD-Arabitol ? − ? − α-Methyl-D- − − − − L-Arabitol − − − − glucosideGluconic acid ? + ? + N-Acetyl- + + + + 2-Ketogluconate − − − −glucosamine 5-Ketogluconate − − − − Amygdalin + + + + Arbutin + + + +

TABLE 2 APsulloc KCTC3108 331263 Substrate 24 h 48 h 24 h 48 h Control −− − − Glycerol − − − − Erythritol − − − − D-Arabinose − − − −L-Arabinose + + + + Ribose + + + + D-Xylose − − − − L-Xylose − − − −Adonitol − − − − β-Methyl- − − − − D-xylose Galactose + + + +Glucose + + + + Fructose + + + + Mannose + + + + Sorbose − − − −Rhamnose − − − − Dulcitol − − − − Inositol − − − − Mannitol + + + +Sorbitol + + + + α-Methyl-D- ? + − − mannoside α-Methyl-D- − − − −glucoside N-Acetyl- + + + + glucosamine Amygdalin + + + +Arbutin + + + + Esculin + + + + Salicin + + + + Cellobiose ? + + +Maltose + + + + Lactose + + + + Melibiose + + + + D-Saccharose + + + +(Sucrose) Trehalose + + + + Inulin − − − − Melezitose + + + + Raffinose− − ? + Amidon − − − − (Starch) Glycogen − − − − Xylitol − − − −Gentiobiose − − + + D-Turanose + + − − D-Lyxose − − − − D-Tagatose − − −− D-Fucose − − − − L-Fucose − − − − D-Arabitol ? − − − L-Arabitol − − −− Gluconic acid ? + + + 2-Ketogluconate − − − − 5-Ketogluconate − − − −

TABLE 3 APsulloc KCTC3108 331266 Substrate 24 h 48 h 24 h 48 h Control −− − − Glycerol − − − − Erythritol − − − − D-Arabinose − − − −L-Arabinose + + + + Ribose + + + + D-Xylose − − − − L-Xylose − − − −Adonitol − − − − β-Methyl- − − − − D-xylose Galactose + + + +Glucose + + + + Fructose + + + + Mannose + + + + Sorbose − − − −Rhamnose − − − − Dulcitol − − − − Inositol − − − − Mannitol + + + +Sorbitol + + + + α-Methyl-D- ? + − − Mannoside α-Methyl-D- − − − −glucoside N-Acetyl- + + + + glucosamine Amygdalin + + + +Arbutin + + + + Esculin + + + + Salicin + + + + Cellobiose ? + + +Maltose + + + + Lactose + + + + Melibiose + + + + D-Saccharose + + + +(Sucrose) Trehalose + + + + Inulin − − − − Melezitose + + + + Raffinose− − + + Amidon − − − − (Starch) Glycogen − − − − Xylitol − − − −Gentiobiose − − + + D-Turanose + + + + D-Lyxose − − − − D-Tagatose − − −− D-Fucose − − − − L-Fucose − − − − D-Arabitol ? − − − L-Arabitol − − −− Gluconic acid ? + + + 2-Ketogluconate − − − − 5-Ketogluconate − − − −

TABLE 4 APsulloc KCTC3108 331269 Substrate 24 h 48 h 24 h 48 h Control −− − − Glycerol − − − − Erythritol − − − − D-Arabinose − − − −L-Arabinose + + − − Ribose + + + + D-Xylose − − + + L-Xylose − − − −Adonitol − − − − β-Methyl- − − − − D-xylose Galactose + + + +Glucose + + + + Fructose + + + + Mannose + + + + Sorbose − − − −Rhamnose − − − − Dulcitol − − − − Inositol − − − − Mannitol + + + +Sorbitol + + + + α-Methyl-D- ? + ? + mannoside α-Methyl-D- − − − −glucoside N-Acetyl- + + + + glucosamine Amygdalin + + + +Arbutin + + + + Esculin + + + + Salicin + + + + Cellobiose ? + + +Maltose + + + + Lactose + + + + Melibiose + + + + D-Saccharose + + + +(Sucrose) Trehalose + + + + Inulin − − − − Melezitose + + + + Raffinose− − + + Amidon − − − − (Starch) Glycogen − − − − Xylitol − − − −Gentiobiose − − + + D-Turanose + + + + D-Lyxose − − − − D-Tagatose − − −− D-Fucose − − − − L-Fucose − − − − D-Arabitol ? − − − L-Arabitol − − −− Gluconic acid ? + ? + 2-Ketogluconate − − − − 5-Ketogluconate − − − −+: Substrate is degraded, −: Substrate is not degraded, ?: Unable todetermine

TABLE 5 Strain Name % Index T Index KCTC3108 Lactobacillus plantarum99.9 0.8 Lactobacillus pentosus 0.1 0.29 APsulloc Lactobacillusplantarum 99.4 0.99 331261 Lactobacillus pentosus 0.4 0.71 APsullocLactobacillus plantarum 98.9 0.97 331263 Lactobacillus pentosus 0.7 0.71APsulloc Lactobacillus plantarum 99.4 0.99 331266 Lactobacillus pentosus0.4 0.71 APsulloc Lactobacillus plantarum 99.4 0.79 331269 Lactobacilluspentosus 0.5 0.51

As can be seen from the above, all of the APsulloc 331261, the APsulloc331263, the APsulloc 331266 and the APsulloc 331269 show the consistency(% index) to the Lactobacillus plantarum of 99% or more. Accordingly, itis confirmed that those strains are belong to the Lactobacillusplantarum.

Further, compared with the standard strain (KCTC3108), the APsulloc331261 is different in use of α-methyl-mannoside and raffinose, theAPsulloc 331263 is different in use of α-methyl-mannoside, raffinose andD-turanose, the APsulloc 331266 is different in use ofα-methyl-mannoside and raffinose, and the APsulloc 331269 is differentin use of L-arabinose and raffinose. Accordingly, it is confirmed thatall of them are different strains from the standard strain.

(3) Analysis of Enzyme Activity Pattern of Lactobacillus PlantarumStrain

The APsulloc 331261 strain culture solution prepared as described in (1)is inoculated to MRS broth 10 mL to the concentration of 0.5% andcultured at 37° C. overnight. The culture solution is centrifuged at8,000 rpm for 5 min, supernatant is removed, and then only bacteria arecollected. Then, 0.85% saline buffer 2 mL is added to the bacteria andsuspended. Later process is conducted by using API ZYM kit (Biomerieux)according to a manufacturer's protocol. Specific process is as follows.

First of all, while gradually adding the strain suspension to APIsuspension medium 5 mL, the amount of suspension needed to makecloudiness of about McFarland Standard 2 (Biomerieux) is measured. Twiceof the measured amount of the suspension is added to API 50CHL medium 10mL, and then shaken for mixing. The above mixture 65 μl is added theeach cupule, and cultured at 37° C. for 4 hours. Each one drop of ZYM Areagent and ZYM B reagent is put into each cupule, and 5 min later,scored from 0 to 5 according to the color intensity, and then the score3 or more is decided as positive.

The method described above is repeated to the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 strain culture solution toanalyze enzyme activity patter.

The results of enzyme activity patterns of the APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269, comparedwith Lactobacillus plantarum strain (KCTC3108) as a standard strain areas shown in the following Tables. Table 6: APsulloc 331261, Table 7:APsulloc 331263, Table 8: APsulloc 331266 and Table 9: APsulloc 331269.

TABLE 6 KCTC3108 APsulloc 331261 Enzyme Score Result Score ResultControl 0 − 0 − Alkaline phosphatase 0 − 1 − Esterase 1 − 2 − Esteraselipase 1 − 2 − Lipase 0 − 2 − Leucine arylamidase 5 + 4 + Valinearylamidase 4 + 4 + Cystine arylamidase 1 − 2 − Trypsin 0 − 0 −α-Chymotrypsin 0 − 1 − Acid phosphatease 1 − 3 +Naphthol-AS-BI-phosphohydrolase 1 − 3 + α-Galactosidase 1 − 3 +β-Galactosidase 5 + 5 + β-Glucuronidase 1 − 2 − β-Glucosidase 3 + 5 +N-Acetyl-β-glucosaminidase 0 − 4 + α-Mannosidase 0 − 1 − α-Fucosidase 0− 1 −

TABLE 7 KCTC3108 APsulloc 331263 Enzyme Score Result Score ResultControl 0 − 0 − Alkaline phosphatase 0 − 1 − Esterase 1 − 2 − Esteraselipase 1 − 2 − Lipase 0 − 1 − Leucine arylamidase 5 + 4 + Valinearylamidase 4 + 4 + Cystine arylamidase 1 − 2 − Trypsin 0 − 0 −α-Chymotrypsin 0 − 1 − Acid phosphatease 1 − 3 +Naphthol-AS-BI-phosphohydrolase 1 − 3 + α-galactosidase 1 − 3 +β-galactosidase 5 + 5 + β-glucuronidase 1 − 2 − α-glucosidase 3 + 2 −β-glucosidase 3 + 5 + N-acetyl-β-glucosaminidase 0 − 4 + α-Mannosidase 0− 1 − α-Fucosidase 0 − 1 −

TABLE 8 KCTC3108 APsulloc 331266 Enzyme Score Result Score ResultControl 0 − 0 − Alkaline phosphatase 0 − 1 − Esterase 1 − 1 − Esteraselipase 1 − 1 − Lipase 0 − 1 − Leucine arylamidase 5 + 4 + Valinearylamidase 4 + 3 + Cystine arylamidase 1 − 2 − Trypsin 0 − 1 −α-Chymotrypsin 0 − 1 − Acid phosphatease 1 − 3 +Naphthol-AS-BI-phosphohydrolase 1 − 3 + α-galactosidase 1 − 3 +β-galactosidase 5 + 5 + β-glucuronidase 1 − 2 − β-glucosidase 3 + 5 +N-acetyl-β-glucosaminidase 0 − 4 + α-Mannosidase 0 − 1 + α-Fucosidase 0− 0 −

TABLE 9 KCTC3108 APsulloc 331269 Enzyme Score Result Score ResultControl 0 − 0 − Alkaline phosphatase 0 − 1 − Esterase 1 − 1 − Esteraselipase 1 − 1 − Lipase 0 − 1 − Leucine arylamidase 5 + 4 + Valinearylamidase 4 + 3 + Cystine arylamidase 1 − 2 − Trypsin 0 − 1 −α-Chymotrypsin 0 − 1 − Acid phosphatease 1 − 3 +Naphthol-AS-BI-phosphohydrolase 1 − 3 + α-galactosidase 1 − 3 +β-galactosidase 5 + 5 + β-glucuronidase 1 − 2 − β-glucosidase 3 + 5 +N-acetyl-β-glucosaminidase 0 − 4 + α-Mannosidase 0 − 1 − α-Fucosidase 0− 0 −

As can be seen from the above, all of the APsulloc 331261, the APsulloc331263, the APsulloc 331266 and the APsulloc 331269 have differentenzyme activity intensities of acid phosphatease,naphthol-AS-BI-phosphohydrolase, α-galactosidase andN-acetyl-6-glucosaminidase from the standard strain (KCTC3108).Accordingly, it is confirmed that all of the APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269 aredifferent strains from the standard strain.

Further, the APsulloc 331261, the APsulloc 331263, the APsulloc 331266and the APsulloc 331269 are decided as negative against theβ-glucuronidase activity known as one of representative oncogenicenzymes, which can induce cancer by modifying procarcinogen tocarcinogen in the intestine. In addition, the APsulloc 331263 inhibitsthe α-glucosidase activity. Accordingly, it is confirmed that all of theAPsulloc 331261, the APsulloc 331263, the APsulloc 331266 and theAPsulloc 331269 are acceptable to be used to a food composition.

(4) Evaluation of Antibiotic Resistance of Lactobacillus PlantarumStrain

Sterilized MRS agar 20 mL is added to a petri dish (Diameter: 100 mm),and cooled in a clean bench to prepare a medium. The APsulloc 331261strain culture solution prepared in (1) is inoculated to MRS broth 10 mLto the concentration of 0.5%, and then cultured at 37° C. for 6 hours.The resulting solution is diluted to have absorbance of about 0.08 to0.13 at 625 nm. A sterilized cotton swab is fully soaked in the dilutedsolution, and then streaked evenly on the prepared MRS agar plateoverall. An antibiotic sensitivity disk is dropped on the plate with aproper distance. After culturing at 37° C. for 24 hours, diameter of aclear zone is measured.

The method described above is repeated to the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 strain culture solutions toevaluate antibiotic resistance.

Concentration of the antibiotic sensitivity disk and evaluation standardare as follows.

TABLE 10 Antibiotics Zone Diameter Analysis Ingredient Concen- ResistantIntermediate Susceptible Name tration (R) (I) (S) Ampicillin 10 μg ≦1314-16 ≧17 Ceftazidime 30 μg ≦14 15-17 ≧18 Chloramphenicol 30 μg ≦1213-17 ≧18 Ciprofloxacin 5 μg ≦15 16-20 ≧21 Clindamycin 2 μg ≦14 15-20≧21 Erythromycin 15 ug ≦13 14-22 ≧23 Gentamycin 120 μg ≦6 7-9 ≧10Imipenem 10 μg ≦13 14-15 ≧16 Streptomycin 10 μg ≦11 12-14 ≧15 Neomycin30 μg ≦12 13-16 ≧17 Nitrofurantoin 300 μg ≦14 15-16 ≧17 Penicillin 10 U≦14 — ≧15 Polymyxin B 300 U ≦8  9-11 ≧12 Tetracycline 30 μg ≦14 15-18≧19 Trimethoprim 5 μg ≦10 11-15 ≧16 Vancomycin 30 μg ≦14 15-16 ≧17

The results of antibiotic resistance evaluation of the APsulloc 331261,the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269,compared with Lactobacillus plantarum strain (KCTC3108) as a standardstrain, are as follows. Table 11: APsulloc 331261 and APsulloc 331263,and Table 12: APsulloc 331266 and APsulloc 331269.

TABLE 11 KCTC3108 APsulloc 331261 APsulloc 331263 Zone Zone ZoneAntibiotics (mm) Result (mm) Result (mm) Result Ampicillin 29 S 27 S 31S Ceftazidime 23 S 16 I 15 I Chloramphenicol 24 S 24 S 25 SCiprofloxacin — R — R — R Clindamycin 12 R 29 S 8 R Erythromycin 26 S 25S 27 S Gentamycin 20 S 19 S 19 S Imipenem 39 S 39 S 42 S Streptomycin —R — R — R Neomycin 11 R 9 R 9 R Nitrofurantoin — R 27 S 29 S Penicillin24 S 18 S 19 S Polymyxin B — R — R — R Tetracycline 17 I 17 I 17 ITrimethoprim — R — R — R Vancomycin — R — R — R

TABLE 12 KCTC3108 APsulloc 331266 APsulloc 331269 Zone Zone ZoneAntibiotics (mm) Result (mm) Result (mm) Result Ampicillin 29 S 21 S 21S Ceftazidime 23 S 10 R 10 I Chloramphenicol 24 S 22 S 22 SCiprofloxacin — R — R — R Clindamycin 12 R 9 R 9 S Erythromycin 26 S 28S 28 S Gentamycin 20 S 18 S 18 S Imipenem 39 S 39 S 39 S Streptomycin —R — R — R Neomycin 11 R 9 R 9 R Nitrofurantoin — R 27 S 27 S Penicillin24 S 12 R 12 R Polymyxin B — R — R — R Tetracycline 17 I 17 I 17 ITrimethoprim — R — R — R Vancomycin — R — R — R R: Resistant, I:Intermediate, S: Susceptible

As can be seen from the above, compared with the standard strain(KCTC3108), the APsulloc 331261 is different in antibiotic resistancepattern of ceftazidime, clindamycin and nitrofurantoin, and the APsulloc331263 is different in antibiotic resistance pattern of ceftazidime andnitrofurantoin. Compared with the standard strain (KCTC3108), theAPsulloc 331266 is different in antibiotic resistance pattern ofceftazidime, nitrofurantoin and penicillin, and the APsulloc 331269 isdifferent in antibiotic resistance pattern of ceftazidime, clindamycin,nitrofurantoin and penicillin. Accordingly, it is confirmed that all ofthe APsulloc 331261, the APsulloc 331263, the APsulloc 331266 and theAPsulloc 331269 are different strains from the standard strain.

When analyzing sugar fermentation pattern, enzyme activity pattern andantibiotic resistance pattern overall, it is confirmed that all of theAPsulloc 331261, the APsulloc 331263, the APsulloc 331266 and theAPsulloc 331269 are belong to the Lactobacillus plantarum, and aredifferent strains from the Lactobacillus plantarum standard strain(KCTC3108).

Test Example 1 Acid Resistance Evaluation (1) Acid Resistance EvaluationDepending on pH

The APsulloc 331261 strain culture solution is inoculated to MRS broth10 mL to the concentration of 0.5%, and cultured at 37° C. overnight. pHis controlled to 2.0, 2.5, 3.0, 3.5, respectively, with HCl. The culturesolution 50 μl is inoculated to sterilized MRS broth 5 mL, and thencultured at 37° C. for 1 hour. After 1 hour, the culture solution isdiluted with peptone saline buffer solution to measure the number ofbacteria per mL. After measuring the number of bacteria in the culturesolution, viability is calculated by considering the value multiplyingthe number by 0.01 as control, and considering the number of controlbacteria as 100%.

The method described above is repeated to the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 strain culture solutions toevaluate acid resistance depending on pH.

The results of comparing acid resistance of the APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269 with theLactobacillus plantarum strain (KCTC3108) as a standard strain are shownin the following Tables. Table 13: APsulloc 331261 and APsulloc 331263,and Table 14: APsulloc 331266 and APsulloc 331269.

TABLE 13 KCTC3108 APsulloc 331261 APsulloc 331263 Viability ViabilityViability pH cfu/ml (%) cfu/ml (%) cfu/ml (%) Control 2.9 × 10⁷ 100 3.8× 10⁷ 100 1.9 × 10⁷ 100 2.0 3.0 × 10¹ 0 1.0 × 10¹ 0 <10¹ 0 2.5 2.5 × 10⁷87.4 3.3 × 10⁷ 87.7 1.4 × 10⁷ 76.6 3.0 3.2 × 10⁷ 110.3 3.7 × 10⁷ 110.31.8 × 10⁷ 94.7 3.5 2.8 × 10⁷ 97.7 4.0 × 10⁷ 105.3 1.8 × 10⁷ 93.4

TABLE 14 KCTC3108 APsulloc 331266 APsulloc 331269 Viability ViabilityViability pH cfu/ml (%) cfu/ml (%) cfu/ml (%) Control 2.9 × 10⁷ 100 5.0× 10⁷ 100 5.0 × 10⁷ 100 2.0 3.0 × 10¹ 0 <10¹ 0 3.6 × 10² 0 2.5 2.5 × 10⁷87.4 6.0 × 10⁷ 118.2 5.6 × 10⁷ 130.7 3.0 3.2 × 10⁷ 110.3 — — 6.5 × 10⁷112.7 3.5 2.8 × 10⁷ 97.7 5.8 × 10⁷ 114.2 6.2 × 10⁷ 124.0

As can be seen from the above, all of the APsulloc 331261, the APsulloc331263, the APsulloc 331266 and the APsulloc 331269 show higherviability at about pH 2.5 to 3.5 than the standard strain. Namely, it isconfirmed that the above Lactobacillus plantarum strains have excellentacid resistance. When considering pH of the stomach with foods is about2 to 3, it is confirmed that the Lactobacillus plantarum strains mayhave higher viability in the stomach when contained in the foodcomposition.

(2) Acid Resistance Evaluation Depending on Time

The APsulloc 331261 culture solution is inoculated to MRS broth 10 mL tothe concentration of 0.5%, and cultured at 37° C. overnight. The culturesolution 50 μl, which is controlled to pH 2.5 with HCl, is inoculated tosterilized MRS broth 5 mL, and cultured at 37° C. for 3 hours. Aftereach 1 hour and 3 hours, the culture solution is diluted with peptonesaline buffer solution, and then the number of bacteria per mL ismeasured. After measuring the number of bacteria in the culturesolution, viability is calculated by considering the value multiplyingthe number by 0.01 as control, and considering the number of controlbacteria as 100%.

The method described above is repeated to the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 strain culture solutions toevaluate acid resistance depending on time.

The results of comparing acid resistance of the APsulloc 331261, theAPsulloc 331263, the APsulloc 331266 and the APsulloc 331269 with theLactobacillus plantarum strain (KCTC3108) as a standard strain are shownin the following Tables. Table 15: APsulloc 331261 and APsulloc 331263,and Table 16: APsulloc 331266 and APsulloc 331269.

TABLE 15 KCTC3108 APsulloc 331261 APsulloc 331263 Time ViabilityViability Viability (hr) cfu/ml (%) cfu/ml (%) cfu/ml (%) 0 3.3 × 10⁷100 6.8 × 10⁷ 100 3.4 × 10⁷ 100 1 2.9 × 10⁷ 86.9 6.6 × 10⁷ 98 2.9 × 10⁷87.2 3 2.2 × 10⁷ 67.3 6.4 × 10⁷ 94.6 2.7 × 10⁷ 79.9

TABLE 16 KCTC3108 APsulloc 331266 APsulloc 331269 Time ViabilityViability Viability (hr) cfu/ml (%) cfu/ml (%) cfu/ml (%) 0 3.3 × 10⁷100 6.2 × 10⁷ 100 7.1 × 10⁷ 100 1 2.9 × 10⁷ 86.9 5.7 × 10⁷ 91.4 7.6 ×10⁷ 107.1 3 2.2 × 10⁷ 67.3 5.8 × 10⁷ 92.5 5.6 × 10⁷ 78.8

A can be seen from the above, it is confirmed that all of the APsulloc331261, the APsulloc 331263, the APsulloc 331266 and the APsulloc 331269have higher viability at pH 2.5 even after 1 hour and 3 hours,respectively, than the standard strain. Namely, it is confirmed that allof the above Lactobacillus plantarum strains have excellent acidresistance for a long time. When considering average retention time ofthe intaken foods in the stomach is about 1 to 3 hours, it is confirmedthat the Lactobacillus plantarum strains may have higher viabilityduring the time stayed in the stomach when contained in the foodcomposition.

Thus, all of the APsulloc 331261, the APsulloc 331263, the APsulloc331266 and the APsulloc 331269 are viable even at lower pH of thestomach during the time stayed in the stomach. Accordingly, when thestrains are intaken as a food, those are viable in the stomach, andfurther, have high intestinal deliverity.

Test Example 2 Bile Acid Resistance Evaluation

The APsulloc 331261 culture solution is inoculated to MRS broth 10 mL tothe concentration of 0.5%, and cultured at 37° C. overnight. Ox gall isadded thereto to the concentration of 0.3% and 0.5%, respectively, toprepare a MRS agar plate. MRS agar where the ox gall is not added isused as control. After diluting the strain culture solution andstreaking on MRS agar medium, the plate is cultured at 37° C. for 2days. The results of calculating the number of each colony and theviability (%) of the APsulloc 331261 when considering the number ofcontrol bacteria as 100% are shown in the following Tables. The methoddescribed above is repeated to the APsulloc 331263, the APsulloc 331266and the APsulloc 331269 strain culture solutions to evaluate bile acidresistance, and then the results thereof are shown in the followingTables. Table 17: APsulloc 331261 and APsulloc 331266, and Table 18:APsulloc 331263 and APsulloc 331269.

TABLE 17 APsulloc 331261 Ox Gall Viability APsulloc 331266 Concentration(%) cfu/ml (%) cfu/ml Viability (%) 0 3.3 × 10⁹ 100 3.3 × 10⁹ 100 0.33.2 × 10⁹ 96.5 3.2 × 10⁹ 96.8 0.5 3.0 × 10⁹ 89 3.0 × 10⁹ 85

TABLE 18 Ox Gall Concen- KCTC3108 APsulloc 331263 APsulloc 331269tration Viability Viability Viability (%) cfu/ml (%) cfu/ml (%) cfu/ml(%) 0 3.3 × 10⁹ 100 3.3 × 10⁹ 100 3.3 × 10⁹ 100 0.3 3.2 × 10⁹ 97 3.2 ×10⁹ 111.9 3.2 × 10⁹ 98.1 0.5 3.0 × 10⁹ 91.9 3.0 × 10⁹ 106.9 3.0 × 10⁹95.3

As can be seen from the above, all of the APsulloc 331261, the APsulloc331263, the APsulloc 331266 and the APsulloc 331269 show excellent bileacid resistance. It is known that the strain having excellent bile acidresistance is also excellent in intestinal fix. Accordingly, it isconfirmed that all of the APsulloc 331261, the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 have excellent intestinal fixand intestinal deliverity.

Test Example 3 Lactic Acid Producing Ability Evaluation

The APsulloc 331261 strain culture solution is inoculated to MRS broth10 mL to the concentration of 0.5% and cultured at 37° C. overnight. Theculture solution is centrifuged at 8,000 rpm for 15 min, onlysupernatant is collected. The collected supernatant is treated at 80° C.for 15 min to stop enzyme reaction. The heat treated supernatant isdiluted 100 times with distilled water. Later process is conducted byusing D-lactic acid/L-lactic acid UV method kit (R-biopharm) accordingto a manufacturer's protocol. Specific process is as follows

Kit solution 1 (glycylglycine buffer/L-glutamate) 1 mL, solution 2 (NADsolution) 0.2 mL and GPT suspension solution 3 0.02 mL are sequentiallyadded to a cuvette. The supernatant prepared above 0.1 mL is added tothe cuvette. Deionized water 1 mL is added to control, and deionizedwater 0.9 mL is added to the sample followed by well mixing thereof.After 5 min, absorbance (A1) is measured at 340 nm. D-LDH solution 40.02 mL is added thereto, and mixed well followed by reacting for 30min. Absorbance (A2) is measured at 340 nm. L-LDH solution 5 0.02 mL isadded thereto, and mixed well followed by reacting for 30 min.Absorbance (A3) is measured at 340 nm. The concentrations of D-lacticacid and L-lactic acid in samples are calculated according to acalculating method. The results compared with the Lactobacillusplantarum strain (KCTC3108) as a standard strain are shown in thefollowing Table.

The method described above is repeated to the APsulloc 331263, theAPsulloc 331266 and the APsulloc 331269 strain culture solutions toevaluate lactic acid producing ability, and the results thereof areshown in the following Tables. Table 19: APsulloc 331261 and APsulloc331263, and Table 20: APsulloc 331266 and APsulloc 331269.

TABLE 19 Lactic KCTC3108 APsulloc 331261 APsulloc 331263 acid Conc.Ratio Conc. Ratio Conc. Ratio (g/L) (%) (g/L) (%) (g/L) (%) D-type 11.372 8.9 62 9.2 69 L-type 4.3 28 5.5 38 4.2 31 Total 15.6 100 14.4 10013.4 100

TABLE 20 APsulloc 331269 Lactic KCTC3108 APsulloc 331266 Ratio acidRatio (%) Conc. (g/L) Ratio (%) Conc. (g/L) (%) D-type 72 10.2 62 11.769 L-type 28 6.3 38 5.2 31 Total 100 16.5 100 16.9 100

As can be seen from the above, all of the APsulloc 331263, the APsulloc331266 and the APsulloc 331269 produce D-lactic acid and L-lactic acid.Further, the total lactic acid yields of them are smaller than thestandard strain, and the ratio of the D-lactic acid to the producedlactic acid is low. Accordingly, foods containing the Lactobacillusplantarum strains may be freely taken to adults or infants, who aresusceptible to lactic acid, and when foods are fermented by using theLactobacillus plantarum strains, the foods may have gentler flavor.

Test Example 4 Antibacterial Activity Evaluation (1) APsulloc 331261

The APsulloc 331261 culture solution is inoculated to MRS broth 10 mL tothe concentration of 0.5%, and cultured at 37° C. overnight. SterilizedMRS agar 15 mL is aliquoted to a petri dish to prepare a medium, andthen each 1 μl of the APsulloc 331261 culture solution is spottedthereon, and cultured at 37° C. for 24 hours.

On the other hand, Shigella flexneri is streaked on Tryptic soy agar,and cultured at 37° C. overnight. Then a colony is inoculated to BHIbroth and cultured overnight. BHI soft agar (agar 1%) is sterilized andcooled to about 45 to 50° C., and then Shigella flexneri culturesolution 1% is inoculated thereto.

The Shigella flexneri culture solution 10 mL is overlaid on theAPsulloc331261 culture solution and hardened. After culturing at 37° C.for 24 hours, size of a clear zone is measured. The results comparedwith the Lactobacillus plantarum strain (KCTC3108) as a standard strainare shown in the following Table.

TABLE 21 Test Strain Clear Zone Diameter (mm) KCTC No. Strain KCTC3108APsulloc 331261 2008 Shigella flexneri 26 27

As can be seen from the above, it is confirmed that the APsulloc 331261is more excellent in antibacterial effect against the Shigella flexnerithan the standard strain.

(2) APsulloc 331266

The APsulloc 331266 culture solution is inoculated to MRS broth 10 mL tothe concentration of 0.5%, and cultured at 37° C. overnight.

On the other hand, Listeria monocytogens and Bacillus cereus arestreaked on Tryptic soy agar, respectively, and cultured at 37° C.overnight. Then a colony is inoculated to BHI broth and culturedovernight.

BHI soft agar (agar 1%) is sterilized and cooled to about 45 to 50° C.,and then Listeria monocytogens and Bacillus cereus culture solutions 1%are inoculated thereto, respectively. 15 mL of the resulting solutionsare aliquoted to a petri dish, respectively, and cooled about 1 hour toprepare a medium. 5 mL of sterilized MRS soft agar (agar 1%) is overlaidon the medium prepared above and hardened. Each 5 μl of the APsulloc331266 culture solution is spotted on the hardened medium and dried.After culturing at 37° C. for 24 hours, size of a clear zone ismeasured. The results compared with the Lactobacillus plantarum strain(KCTC3108) as a standard strain are shown in the following Table.

TABLE 22 Test Strain Clear Zone Diameter (mm) KCTC no. strain KCTC3108APsulloc 331266 3710 Listeria monocytogens 16.8 19.3 3624 Bacilluscereus 10.3 12.3

As can be seen from the above, it is confirmed that the APsulloc 331266is more excellent in antibacterial effect against the Listeriamonocytogens and the Bacillus cereus than the standard strain.

(3) APsulloc 331269

The APsulloc 331269 culture solution is inoculated to MRS broth 10 mL tothe concentration of 0.5%, and cultured at 37° C. overnight. The culturesolution is centrifuged at 8000 rpm for 5 min, and only supernatant iscollected. The supernatant 10 mL is prepared by sterilizing with a 0.22μl syringe filter.

On the other hand, Salmonella typhimurium and Staphylococcus aureus arestreaked on Tryptic soy agar, respectively, and cultured at 37° C.overnight. Then a colony is inoculated to BHI broth and culturedovernight.

BHI agar is sterilized, and 20 mL thereof is put into a petri dish andcooled to prepare a medium. Salmonella typhimurium and Staphylococcusaureus culture solutions are diluted 50 times with sterilizedphysiological saline solution, respectively, and then streaked evenly onthe medium prepared above with a sterilized cotton swab. A sterilizedpaper disk is put thereon, and the supernatant of the APsulloc 331269culture solution prepared above 100 μl is dropped on the paper disk. Themedium is stored at room temperature for about 3 hours for absorption,and then cultured at 37° C. for 24 hours. Size of a clear zone ismeasured, and the results compared with the Lactobacillus plantarumstrain (KCTC3108) as a standard strain are shown in the following Table.

TABLE 23 Test Strain Clear Zone Diameter (mm) KCTC No. Strain KCTC3108APsulloc 331269 2514 Salmonella typhimurium 8.8 9.3 1621 Staphylococcusaureus 10 10.8

As can be seen from the above, it is confirmed that the APsulloc 331269is more excellent in antibacterial effect against the Salmonellatyphimurium and the Staphylococcus aureus than the standard strain.

1. A method of treating an intestinal disorder, said method comprisingadministering an effective dose of Lactobacillus plantarum APsulloc331263 (Accession No. KCCM11180P) strain, an extract thereof, or aculture solution thereof to a subject in need of treating an intestinaldisorder, wherein the strain has acid resistance at pH 2 to pH 4, andwherein the strain has bile resistance at 0.3-0.5% bile concentration.2. The method according to claim 1, wherein the intestinal disordercomprises one or more of irritable bowel syndrome, constipation, anddiarrhea.
 3. The method according to claim 1, wherein the strain, theextract thereof, or the culture solution thereof is administered in acomposition, wherein the composition further comprises at least one of asurfactant, excipient, wetting agent, emulsifier, suspending agent, saltor buffer for osmotic pressure control, colorant, flavoring agent,stabilizer, preservative, and preserved agent.
 4. The method accordingto claim 1, wherein the strain is isolated from tea tree (Camelliasinensis) leaves.
 5. The method according to claim 1, wherein the strainhas acid resistance at pH 2 to pH 4 for 0.5 hr to 5 hours.
 6. The methodaccording to claim 1, wherein the strain has lactic acid producingability.
 7. The method according to claim 6, wherein the strain haslactic acid producing ability, which produces D-type lactic acid in anamount of 70% or less based on a total amount of produced lactic acid.8. The method of claim 1, wherein the effective dose is 0.1 mg/kg/day to5000 mg/kg/day.
 9. The method of claim 1, wherein administration is oralor parenteral.